The present invention is in the field of nuclear hormone receptor proteins that are related to the retinoic acid receptor nuclear hormone receptor subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel nuclear hormone receptor peptides and proteins and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
Nuclear Hormone Receptors
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. The biochemical pathways through which signals are transmitted within cells comprise a circuitry of directly or functionally connected interactive proteins. One of the key biochemical mechanisms of signal transduction involves the activation of gene transcription. Many of the transcription activation pathways are controlled by the action of intracellular receptors (IRs), such as members of the nuclear hormone receptor family of proteins and their ligands. The binding of a ligand to a nuclear hormone receptor serves to translate signals generated from a variety of cellular events.
Intracellular receptors (IRs) form a class of structurally related genetic regulators scientists have named xe2x80x9cligand dependent transcription factors.xe2x80x9d R. M. Evans, 240 Science, 889 (1988). Nuclear hormone receptors are a recognized subset of the IRs, including the progesterone receptor (PR), androgen receptor (AR), estrogen receptor (ER), glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). Regulation of a gene by such factors requires both the IR itself and a corresponding ligand which has the ability to selectively bind to the IR in a way that alters gene transcription.
Ligands to the IRs can include low molecular weight native molecules, such as the hormones progesterone, estrogen and testosterone, as well as synthetic derivative compounds such as medroxyprogesterone acetate, diethylstilbesterol and 19-nortestosterone. These ligands, when present in the fluid surrounding a cell, pass through the outer cell membrane by passive diffusion and bind to specific IR proteins to create a ligand/receptor complex. This complex then translocates to the cell""s nucleus, where it binds to a specific gene or genes present in the cell""s DNA. Once bound to DNA, the complex modulates the production of the protein encoded by that gene. In this regard, a compound which binds and IR and mimics the effect of the native ligand is referred to as an xe2x80x9cagonistxe2x80x9d, while a compound that inhibits the effect of the native ligand is called an xe2x80x9cantagonist.xe2x80x9d
Ligands to the nuclear hormone receptors are known to play an important role in health of both women and men. For example, the native female ligand, progesterone, as well as synthetic analogues, such as norgestrel (18-homonorethisterone) and norethisterone (17.alpha.-ethinyl-19-nortestosterone), are used in birth control formulations, typically in combination with the female hormone estrogen or synthetic estrogen analogues, as effective modulators of both PR and ER. On the other hand, antagonists to PR are potentially useful in treating chronic disorders, such as certain hormone dependent cancers of the breast, ovaries, and uterus, and in treating non-malignant conditions such as uterine fibroids and endometriosis, a leading cause of infertility in women. Similarly, AR antagonists, such as cyproterone acetate and flutamide have proved useful in the treatment of hyperplasia and cancer of the prostate.
Nuclear hormone hormones, one sub-family of IRs, are potent modulators of transcriptional events that together regulate the complex processes associated with differentiation homeostasis and development. The mechanism of action of these molecules is related in that the effector molecule binds to a specific intracellular receptor. This binding alters the structure of the receptor, thus increasing its affinity for specific recognition sites within the regulatory region of target genes. In this way, the nuclear hormone directs a program of events that leads to a change in cell phenotype.
Nuclear hormone hormones, thyroid hormones and certain vitamins can regulate cellular differentiation morphogenesis and homeostasis by binding to specific intracellular receptor proteins. Ligand activated receptor complexes are capable of activating or repressing transcription of a specific set of target genes. Thus, the receptor proteins are capable of reprogramming cellular function at the genomic level in response to hormonal or vitamin signals.
Retinoic Acids and Retinoic Acid Receptors
The protein provided by the present invention is a novel retinoic acid receptor isoform; the new isoform provided herein is an ortholog of the rat retinoic acid receptor alpha 2. Consequently, the new retinoic acid receptor isoform provided by the present invention may be named human retinoic acid receptor alpha 2. As used herein, the term retinoic acid (RA) is synonymous with retinoid, and the term retinoic acid receptor is synonymous with retinoid receptor.
Retinoids, or vitamin A metabolites/derivatives, have been determined to play essential roles in many aspects of development, metabolism and reproduction in vertebrates (see, for example, The Retinoids, Second Edition, Sporn et al. (Raven Press, New York, 1994)). There are two classes of retinoid receptors: the retinoic acid receptors (RARs), which bind to both all-trans retinoic acid (atRA) and 9-cis retinoic acid (9cRA), and the retinoid X receptors (RXRs), which bind only to 9cRA. These receptors modulate ligand-dependent gene expression by interacting as RXR/RAR heterodimers or RXR homodimers on specific target gene DNA sequences known as hormone response elements. In addition to their role in retinoid signalling, RXRs also serve as heterodimeric partners of nuclear receptors for vitamin D, thyroid hormone, and peroxisome proliferators (reviewed by Mangelsdorf et al., at pages 319-349 of The Retinoids, Second Edition, Spom et al. (Raven Press, New York, 1994)).
A number of studies have demonstrated that retinoids are essential for normal growth, vision, tissue homeostasis, reproduction and overall survival (for reviews and references, See Sporn et al., The Retinoids, Vols. 1 and 2, Sporn et al., eds., Academic Press, Orlando, Fla. (1984)). For example, retinoids have been shown to be vital to the maintenance of skin homeostasis and barrier function in mammals (Fisher, G. J., and Voorhees, J. J., FASEB J. 10:1002-1013 (1996)). Retinoids are also apparently crucial during embryogenesis, since offspring of dams with vitamin A deficiency (VAD) exhibit a number of developmental defects (Wilson, J. G., et al., Am. J. Anat. 92:189-217 (1953); Morriss-Kay, G. M., and Sokolova, N., FASEB J. 10:961-968 (1996)). With the exceptions of those on vision (Wald, G., et al., Science 162:230-239 (1968)) and spermatogenesis in mammals (van Pelt, H. M. M., and De Rooij, D. G., Endocrinology 128:697-704 (1991)), most of the effects generated by VAD in animals and their fetuses can be prevented and/or reversed by retinoic acid (RA) administration (Wilson, J. G., et al., Am. J. Anat. 92:189-217 (1953); Thompson et al., Proc. Royal Soc. 159:510-535 (1964); Morriss-Kay, G. M., and Sokolova, N., FASEB J. 10:961-968 (1996)). The dramatic teratogenic effects of maternal RA administration on mammalian embryos (Shenefelt, R. E., Teratology 5, 103-108 (1972); Kessel, M., Development 115:487-501 (1992); Creech Kraft, J., In Retinoids in Normal Development and Teratogenesis, G. M. Morriss-Kay, ed., Oxford University Press, Oxford, UK, pp. 267-280 (1992)), and the marked effects of topical administration of retinoids on embryonic development of vertebrates and limb regeneration in amphibians (Mohanty-Hejmadi et al., Nature 355:352-353 (1992); Tabin, C. J., Cell 66:199-217 (1991)), have contributed to the notion that RA may have critical roles in morphogenesis and organogenesis.
Many synthetic structural analogues of all-trans retinoic acid or 9-cis-retinoic acid, commonly termed xe2x80x9cretinoidsxe2x80x9d, have been described in the literature to date. Some of these molecules are able to bind to, and specifically activate, the RARs or, on the other hand, the RXRs. Furthermore, some analogues are able to bind to, and activate a particular RAR receptor subtype (.alpha., .beta. or .gamma.). Finally, other analogues do not exhibit any particular selective activity with regard to these different receptors. In this respect, and by way of example, 9-cis-retinoic acid activates the RARs and the RXRs at one and the same time without any noteworthy selectivity for either of these receptors (nonspecific agonist ligand), whereas all-trans retinoic acid selectively activates the RARs (RAR-specific agonist ligand), with all subtypes being included. In a general manner, and qualitatively, a given substance (or ligand) is said to be specific for a given family of receptors (or, respectively, for a particular receptor of this family) when the said substance exhibits an affinity for all the receptors of this family (or, respectively, for the particular receptor of this family) which is stronger than that which it otherwise exhibits for all the receptors of any other family (or, respectively, for all the other receptors, of this same family or not).
The genetic activities of the RA signal are mediated through the two families of receptorsxe2x80x94the RAR family and the RXR familyxe2x80x94which belong to the superfamily of ligand-inducible transcriptional regulatory factors that include steroid/thyroid hormone and vitamin D3 receptors (for reviews see Leid et al., TIBS 17:427-433 (1992); Chambon, P., Semin. Cell Biol. 5:115-125 (1994); Chambon, P., FASEB J. 10:940-954 (1996); Giguere, V., Endocrinol. Rev. 15:61-79 (1994); Mangelsdorf, D. J., and Evans, R. M., Cell 83:841-850 (1995); Gronemeyer, H., and Laudet, V., Protein Profile 2:1173-1236 (1995)).
RARs are the critical factors in tissue differentiation and development They are up-regulated in rapidly dividing cells and tumors. RARs play an important role in lymphocyte activation. Synthetic antagonists of retinoic acid receptors can inhibit delayed type hypersensitivity (DTH). Growth factors and carotene regulate RXR expression levels. For example, granulocyte macrophage colony-stimulating factor induces retinoic acid receptors in myeloid leukemia cells.
Retinoic acid receptors can form heterodimers with other nuclear receptors. The protein provided by the present invention can be used as a probe to detect possible interactions in the two-hybrid assay. Synthetic peptides that mimic dimerization surface can disrupt intermolecular interactions between these receptors. RAR gene rearrangements are the primary causes of some types of leukemia and provide a convenient genetic marker for malignant cell lines. A number of retinoic acid derivatives are used in treatment of myelodysplastic disorders. They are designed to bind and activate RXRs. Beta-cotene can prevent skin tumor formation in mouse models. N-(4-hydroxyphenyl)retinarmide can delay onset of dysplasia in bronchi. Different chemopreventive drugs can be designed to target individual retinoic receptors. The sequences provided by the present invention may be used to design high affinity chemopreventive compounds.
Although both the RARs and RXRs respond to all-trans-retinoic acid in vivo, the receptors differ in several important aspects. First, the RARs and RXRs are significantly divergent in primary structure (e.g., the ligand binding domains of RAR.alpha and RXR.alpha. have only 27% amino acid identity). These structural differences are reflected in the different relative degrees of responsiveness of RARs and RXRs to various vitamin A metabolites and synthetic retinoids. In addition, distinctly different patterns of tissue distribution are seen for RARs and RXRs. For example, in contrast to the RARS, which are generally not expressed at high levels in the visceral tissues, RXR.alpha. mRNA has been shown to be most abundant in the liver, kidney, lung, muscle and intestine. Finally, the RARs and RXRs have different target gene specificity. For example, response elements have recently been identified in the cellular retinal binding protein type II (CRBPII) and apolipoprotein A1 genes which confer responsiveness to RXR, but not RAR. Furthermore, RAR has also been recently shown to repress RXR-mediated activation through the CRBPII RXR response element (Manglesdorf et al., Cell, 66:555-61 (1991)). These data indicate that two retinoic acid responsive pathways are not simply redundant, but instead manifest a complex interplay. Recently, Heyman et al. (Cell, 68:397-406 (1992)) and Levin et al. (Nature, 355:359-61 (1992)) independently demonstrated that 9-cis-retinoic acid is a natural endogenous ligand for the RXRs. 9-cis-retinoic acid was shown to bind and transactivate the RXRs, as well as the RARs, and therefore appears to act as a xe2x80x9cbifunctionalxe2x80x9d ligand.
RAR Receptors
Receptors belonging to the RAR family (RAR.alpha., .beta. and .gamma. and their isoforms) are activated by both all-trans- and 9-cis-RA (Leid et al., TIBS 17:427-433 (1992); Chambon, P., Semin. Cell Biol. 5:115-125 (1994); Dolle, P., et al., Mech. Dev. 45:91-104 (1994); Chambon, P., FASEB J. 10:940-954 (1996)). Within a given species, the DNA binding (C) and the ligand binding (E) domains of the three RAR types are highly similar, whereas the C-terminal domain F and the middle domain D exhibit no or little similarity. The amino acid sequences of the three RAR types are also notably different in their B regions, and their main isoforms (.alpha.1 and .alpha.2, .beta.1 to .beta.4, and .gamma.1 and .gamma.2) further differ in their N-terminal A regions (Leid et al., TIBS 17:427-433 (1992)). Amino acid sequence comparisons have revealed that the interspecies conservation of a given RAR type is greater than the similarity found between the three RAR types within a given species (Leid et al., TIBS 17:427-433 (1992)). This interspecies conservation is particularly striking in the N-terminal A regions of the various RAR.alpha., .beta. and .gamma. isoforms, whose A region amino acid sequences are quite divergent. Taken together with the distinct spatio-temporal expression patterns observed for the transcripts of each RAR and RXR type in the developing embryo and in various adult mouse tissues (Zelent, A., et al., Nature 339:714-717 (1989); Dolle, P., et al., Nature 342:702-705 (1989); Dolleet al., Development 110:1133-1151 (1990); Ruberte et al., Development 108:213-222 (1990); Ruberte et al., Development 111:45-60 (1991); Mangelsdorf et al., Genes and Dev. 6:329-344 (1992)), this interspecies conservation has suggested that each RAR type (and isoform) may perform unique functions. This hypothesis is further supported by the finding that the various RAR isoforms contain two transcriptional activation functions (AFs) located in the N-terminal A/B region (AF-1) and in the C-terminal E region (AF-2), which can synergistically, and to some extent differentially, activate various RA-responsive promoters (Leid et al., TIBS 17:427433 (1992); Nagpal, S., et al., Cell 70:1007-1019 (1992); Nagpal, S., et al., EMBO J. 12:2349-2360 (1993)).
RXR Receptors
Unlike the RARs, members of the retinoid X receptor family (RXR.alpha., .beta. and .gamma.) are activated exclusively by 9-cis-RA (Chambon, P., FASEB J. 10:940-954 (1996); Chambon, P., Semin. Cell Biol. 5:115-125 (1994); Dolle, P., et al., Mech. Dev. 45:91-104 (1994); Linney, E., Current Topics in Dev. Biol. 27:309-350 (1992); Leid et al., TIBS 17:427-433 (1992); Kastner et al., in Vitamin A in Health and Disease, R. Blomhoff, ed., Marcel Dekker, New York (1993)). However, the RXRs characterized to date are similar to the RARs in that the different RXR types also differ markedly in their N-terminal A/B regions (Leid et al., TIBS 17:427-433 (1992); Leid et al., Cell 68:377-395 (1992); Mangelsdorf et al., Genes and Dev. 6:329-344 (1992)), and contain the same transcriptional activation functions in their N-terminal A/B region and C-terminal E region (Leid et al., TIBS 17:427-433 (1992); Nagpal, S., et al., Cell 70:1007-1019 (1992); Nagpal, S., et al., EMBO J. 12:2349-2360(1993)).
RXR.alpha. and RXR.beta. have a widespread (possibly ubiquitous) expression pattern during mouse development and in the adult animal, being found in all fetal and adult tissues thus far examined (Mangelsdorf, D. J., et al., Genes and Devel. 6:329-344 (1992); Dolle, P., et al., Mech. Devel. 45:91-104 (1994); Nagata, T., et al., Gene 142:183-189 (1994)). RXR.gamma. transcripts, however, appear to have a more restricted distribution, being expressed in developing skeletal muscle in the embryo (where their expression persists throughout life), in the heart (after birth), in sensory epithelia of the visual and auditory systems, in specific structures of the central nervous system, and in tissues involved in thyroid hormone homeostasis, e.g., the thyroid gland and thyrotrope cells in the pituitary (Mangelsdorf, D. J., et al., Genes and Devel. 6:329-344 (1992); Dolle, P., et al., Mech. Devel. 45:91-104 (1994); Sugawara, A., et al., Endocrinology 136:1766-1774 (1995); Liu, Q., and Linney, E., Mol. Endocrinol. 7:651-658 (1993)).
It is currently unclear whether all the molecular properties of RXRs characterized in vitro are relevant for their physiological functions in vivo. In particular, it is unknown under what conditions these receptors act as 9-cis-RA-dependent transcriptional regulators (Chambon, P., Semin. Cell Biol. 5:115-125 (1994)). The knock-outs of RXR.alpha. and RXR.beta. in the mouse have provided some insight into the physiological functions of these receptors. For example, the ocular and cardiac malformations observed in RXR.alpha . . . sup.xe2x88x92/xe2x88x92 fetuses (Kastner, P., et al., Cell 78:987-1003 (1994); Sucov, H. M., et al., Genes and Devel. 8:1007-1018 (1994)) are similar to those found in the fetal VAD syndrome, thus suggesting an important function of RXR.alpha. in the transduction of a retinoid signal during development. The involvement of RXRs in retinoid signaling is further supported by studies of compound RXR.alpha./RAR mutants, which reveal defects that are either absent or less severe in the single mutants (Kastner, P., et al., Cell 78:987-1003 (1994); Kastner, P., et al., Cell 83:859-869 (1995)). Interestingly, however, knockout of RXR.gamma. in the mouse induces no overt deleterious effects, and RXR.gamma . . . sup.xe2x88x92/xe2x88x92 homozygotes which are also RXR.alpha . . . sup.xe2x88x92/xe2x88x92 or RXR.beta . . . sup.xe2x88x92/xe2x88x92 exhibit no additional abnormalities beyond those seen in RXR.alpha . . . sup.xe2x88x92/xe2x88x92, RXR.beta . . . sup.xe2x88x92/xe2x88x92 and fetal VAD syndrome fetuses (Krezel, W., et al., Proc. Natl. Acad. Sci. USA 93(17):9010-9014 (1996)), suggesting that RXR.gamma., despite its highly tissue-specific expression pattern in the developing embryo, is dispensable for embryonic development and postnatal life in the mouse. The observation that live-born RXR.gamma . . . sup.xe2x88x92/xe2x88x92/RXR.beta . . . sup.xe2x88x92/xe2x88x92/RXR.alpha . . . sup.xe2x88x92/xe2x88x92 mutants can grow to reach adult age (Krezel et al., Proc. Natl. Acad. Sci. USA 93(17):9010-9014 (1996)) indicates that a single RXR.alpha. allele is sufficient to carry out all of the vital developmental and postnatal functions of the RXR family of receptors, particularly all of the developmental functions which depend on RARs and may require RXR partnership (Dolle, P., et al., Mech. Dev. 45:91-104 (1994); Kastner, P., et al., Cell 83:859-869 (1995)). Furthermore, the finding that RXR.alpha . . . sup.xe2x88x92/xe2x88x92/RXR.gamma . . . sup.xe2x88x92/xe2x88x92 double mutant embryos are not more affected than are single RXR.alpha . . . sup.xe2x88x92/xe2x88x92 mutants (Krezel et al., Proc. Natl. Acad. Sci. USA 93(17):9010-9014 (1996)) clearly shows that RXR.beta.alone can also perform some of these functions. Therefore, the fact that RXR.alpha. alone and, to a certain extent RXR.beta. alone, are sufficient for the completion of a number of developmental RXR functions, clearly indicates the existence of a large degree of functional redundancy amongst RXRs. In this respect, the RXR situation is different from that of RARs, since all of types of RAR double mutants displayed much broader sets of defects than single mutants Rowe, A., et al., Develop. 111:771-778 (1991); Lohnes, D., et al., Develop. 120:2723-2748 (1994); Mendelsohn, C., Develop. 120:2749-2771 (1994)).
Retinoid Binding to RAR and RXR Receptors
The crystal structures of the ligand-binding domains (LBDs) of the RARs and RXRs have recently been elucidated (Bourget, W., et al., Nature 375:377-382 (1995); Renaud, J. P., et al., Nature 378:681-689 (1995); Wurtz, J. M., et al., Nature Struct. Biol. 3:87-94 (1996)). Among the various RAR types, substantial amino acid sequence identity is observed in these domains: comparison of the LBDs of RAR.alpha., RAR.beta. and RAR.gamma. indicates that only three amino acid residues are variable in the ligand-binding pocket of these receptors. These residues apparently account for the fact that the various RAR types exhibit some selectivity in binding certain synthetic retinoids (Chen, J.-Y., et al., EMBO J. 14(6):1187-1197 (1995); Renaud, J. P., et al., Nature 378:681-689 (1995)), and consideration of these divergent residues can be used to design RAR type-specific synthetic retinoids which may be agonistic or antagonistic (Chambon, P., FASEB J. 10:940-954 (1996)). This design approach may be extendable generally to other nuclear receptors, such as thyroid receptor .alpha. (Wagner, R. L., et al., Nature 378:690-697 (1995)), the ligand-binding pockets of which may chemically and structurally resemble those of the RARs (Chambon, P., FASEB J. 10:940-954 (1996)). Conversely, molecular modeling of the ligand-binding pocket of the RXRs demonstrates that there are no overt differences in amino acid composition between RXR.alpha., RXR.beta. and RXR.gamma. (Bourguet, W., et al., Nature 375:377-382 (1995); Wurtz, J. M., et al., Nature Struct. Biol. 3:87-94 (1996)), suggesting that design of type-specific synthetic ligands for the RXRs may be more difficult than for the RARs (Chambon, P., FASEB J. 10:940-954 (1996)).
Retinoid Signaling Through RAR:RXR Heterodimers
Nuclear receptors (NRs) are members of a superfamily of ligand-inducible transcriptional regulatory factors that include receptors for steroid hormones, thyroid hormones, vitamin D3 and retinoids (Leid, M., et al., Trends Biochem. Sci. 17:427433 (1992); Leid, M., et al., Cell 68:377-395 (1992); and Linney, E. Curr. Top. Dev. Biol., 27:309-350 (1992)). NRs exhibit a modular structure which reflects the existence of several autonomous functional domains. Based on amino acid sequence similarity between the chicken estrogen receptor, the human estrogen and glucocorticoid receptors, and the v-erb-A oncogene (Krust, A., et al., EMBO J. 5:891-897 (1986)), defined six regionsxe2x80x94A, B, C, D, E and Fxe2x80x94which display different degrees of evolutionary conservation amongst various members of the nuclear receptor superfamily. The highly conserved region C contains two zinc fingers and corresponds to the core of the DNA-binding domain (DBD), which is responsible for specific recognition of the cognate response elements. Region E is functionally complex, since in addition to the ligand-binding domain (LBD), it contains a ligand-dependent activation function (AF-2) and a dimerization interface. An autonomous transcriptional activation function (AF-1) is present in the non-conserved N-terminal A/B regions of the steroid receptors. Interestingly, both AF-1 and AF-2 of steroid receptors exhibit differential transcriptional activation properties which appear to be both cell type and promoter context specific (Gronemeyer, H. Annu. Rev. Genet. 25:89-123 (1991)).
As described above, the all-trans (T-RA) and 9-cis (9C-RA) retinoic acid signals are transduced by two families of nuclear receptors, RAR .alpha., .beta. and .gamma. (and their isoforms) are activated by both T-RA and 9C-RA, whereas RXR .alpha., .beta. and .gamma. are exclusively activated by 9C-RA (Allenby, G. et al., Proc. Natl. Acad. Sci. USA 90:30-34 (1993)). The three RAR types differ in their B regions, and their main isoforms (.alpha.1 and .alpha.2, .beta. 1-4, and .gamma.1 and .gamma.2) have different N-terminal A regions (Leid, M. et al., Trends Biochem. Sci. 17:427-433 (1992)). Similarly, the RXR types differ in their A/B regions (Mangelsdorf, D. J. et al., Genes Dev. 6:329-344 (1992)).
The E-region of RARs and RXRs has also been shown to contain a dimerization interface (Yu, V. C. et al., Curr. Opin. Biotechnol. 3:597-602 (1992)). Most interestingly, it was demonstrated that RAR/RXR heterodimers bind much more efficiently in vitro than homodimers of either receptor to a number of RA response elements (RAREs) (Yu, V. C. et al., Cell 67:1251-1266 (1991); Berrodin, T. J. et al., Mol. Endocrinol 6:1468-1478 (1992); Bugge, T. H. et al., EMBO J. 11:1409-1418 (1992); Hall, R. K. et al., Mol. Cell. Biol. 12: 5527-5535 (1992); Hallenbeck P. L. et al., Proc. Natl. Acad. Sci. USA 89:5572-5576 (1992); Husmann, M. et al., Biochem. Biophys. Res. Commun. 187:1558-1564 (1992); Kliewer, S. A. et al., Nature 355:446-449 (1992); Leid, M. et al., Cell 68:377-395 (1992); Marks, M. S. et al., EMBO J. 11:1419-1435 (1992); Zhang, X. K. et al., Nature 355:441-446 (1992)). RAR and RXR heterodimers are also preferentially formed in solution in vitro (Yu, V. C. et al., Cell 67:1251-1266 (1991); Leid, M. et al., Cell 68:377-395 (1992); Marks, M. S. et al., EMBO J. 11:1419-1435 (1992)), although the addition of 9C-RA appears to enhance the formation of RXR homodimers in vitro (Lehman, J. M. et al., Science 258:1944-1946 (1992); Zhang, X. K. et al., Nature 358:587-591 (1992b)).
It has been shown that activation of RA-responsive promoters likely occurs through RAR:RXR heterodimers rather than through homodimers (Yu, V. C. et al., Cell 67:1251-1266 (1991); Leid et al., Cell 68:377-395 (1992b); Durand et al., Cell 71:73-85 (1992); Nagpal et al., Cell 70:1007-1019 (1992); Zhang, X. K., et al., Nature 355, 441-446 (1992); Kliewer et al., Nature 355:446-449 (1992); Bugge et al., EMBO J. 11: 1409-1418 (1992); Marks et al., EMBO J. 11:1419-1435 (1992); Yu, V. C. et al., Cur. Op. Biotech. 3:597-602 (1992); Leid et al., TIBS 17:427-433 (1992); Laudet and Stehelin, Curr. Biol. 2:293-295 (1992); Green, S., Nature 361:590-591 (1993)). The RXR portion of these heterodimers has been proposed to be silent in retinoid-induced signaling (Kurokawa, R., et al., Nature 371:528-531 (1994); Forman, B. M., et al., Cell 81:541-550 (1995); Mangelsdorf, D. J., and Evans, R. M., Cell 83:835-850 (1995)), although conflicting results have been reported on this issue (Apfel, C. M., et al., J. Biol. Chem. 270(51):30765-30772 (1995); see Chambon, P., FASEB J. 10:940-954 (1996) for review). Although the results of these studies strongly suggest that RAR/RXR heterodimers are indeed fractional units that transduce the RA signal in vivo, it is unclear whether all of the suggested heterodimeric combinations occur in vivo (Chambon, P., Semin. Cell Biol. 5:115-125 (1994)). Thus, the basis for the highly pleiotropic effect of retinoids may reside, at least in part, in the control of different subsets of retinoid-responsive promoters by cell-specifically expressed heterodimeric combinations of RAR:RXR types (and isoforms), whose activity may be in turn regulated by cell-specific levels of all-trans- and 9-cis-RA (Leid et al., TIBS 17:427-433 (1992)).
The RXR receptors may also be involved in RA-independent signaling. For example, the observation of aberrant lipid metabolism in the Sertoli cells of RXR.beta . . . sup.xe2x88x92/xe2x88x92 mutant animals suggests that functional interactions may also occur between RXR.beta. and the peroxisomal proliferator-activated receptor signaling pathway (WO 94/26100; Kastner, P., et al., Genes and Devel. 10:80-92 (1996)).
For a further review of retinoic acid receptors, see: Shimizu et al., Cancer Res 2000 August 15;60(16):4544-9; Ponnamperuma et al., Nutr Cancer 2000;37(1):82-8; Yoshimura et al., J Med Chem 2000 Jul 27;43(15):2929-37; Kurie et al., Clin Cancer Res 2000 Aug;6(8):2973-9; Lee et al., J Biol Chem 2000 Aug 17; and Sainty et al., Blood 2000 Aug 15;96(4):1287-96.
The discovery of a new human nuclear hormone receptor proteins and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of biological processes associated with abnormal or unwanted protein gene activation.
The present invention is based in part on the identification of amino acid sequences of human nuclear hormone receptor peptides and proteins that are related to the retinoic acid receptor nuclear hormone receptor subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate nuclear hormone receptor activity in cells and tissues that express the nuclear hormone receptor. Experimental data as provided in FIG. 1 indicates that nuclear hormone receptors of the present invention are expressed in humans in breast, brain, head-neck tissue, testis, placenta, retina, liver, kidney, HeLa cell tissue, and fetal liver-spleen.